PL209415B1 - Amplification-hybridisation method for detecting and typing human papillomavirus - Google Patents

Abstract

The present invention provides amplification and hybridisation method for detecting and typing human papillomavirus (HPV), and the primers and hybridisation probes used in the method. The invention relates to a concrete part of the HPV genome, which is suitable for designing HPV genus-specific and HPV genotype-specific hybridisation oligonucleotide probes.

Description

The invention relates to an amplification primer mix, its use, a method for detecting one or more HPV genotypes in biological samples, and a kit of reagents for HPV detection and typing.

Multiple papillomavirus sequences have been established, see references cited herein: HPV-6: de Villiers et al., J. Virology, 40 (1981); HPV-11: Dartmann et al., Virology 151, 124-130 (1986); HPV-16: Seedorf et al., Virology 145, 181-185 (1985); HPV-18: Cole and Danos, Journal of Molecular Biology 93, 599-608 (1987); HPV-31: Goldsborough et al., Virology 171, 306-311 (1989); HPV-33:

Cole and Streeck, J. Virology, 58, 991-995 (1986); HPV-54: Favre et al., J. Cancer 45, 40-46 (1990); HPV-56: Lorincz, J. Gen. Virol. 70, 3099 (1989).

Detection and typing of HPV has been described in several publications, but in addition to the Southern method and other hybridization techniques, PCR-based methods are the most widely used techniques, as these methods simultaneously ensure high sensitivity, specificity and flexibility of the assay, which provides greater control over analytical requirements.

The human papillomavirus, a member of the Papillomaviridae family, is an oncogenic DNA virus with a circular genome of 8000 bp. The virus exhibits strong epithelial tropism and proliferates only in differentiated epithelial cells. The papillomavirus is suspected to play an etiological role in a wide variety of human diseases, e.g. various skin diseases such as warts, condylomata and skin cancers, and other conditions such as cervical cancer, anogenital carcinomas, and laryngeal cancer. There is evidence that human papillomavirus strongly correlates with the occurrence of these tumors, and this is even true for precancerous lesions (CIN, VIN, VAIN, PIN, PAIN). HPV can be detected in 99% of cervical cancer patients. This close statistical relationship is probably due to the causal role of HPV in the formation of cervical cancer. Based on epidemiological data, patients with different HPV genotypes do not have the same level of risk of developing cervical cancer. According to these findings, the genotypes have been classified into low-risk, medium-risk and high-risk classes, and there are also unclassified genotypes. Due to the fact that the risk varies widely and the incidence of HPV infection is very high, determination of the genotype is very important.

HPV cannot be grown. Serological diagnosis of HPV infection is limited to the detection of exposure to the virus (past or present infection), but cannot accurately define genotype and its role is mainly limited to epidemiological studies.

For papillomaviruses, there is no exact serological classification (serotyping) and genotyping is a commonly accepted classification method. They can be divided into two groups depending on whether detection is preceded by amplification or not. In one embodiment of the latter method, full-length genomic RNA probes are used to detect HPV genomes in denatured DNA, and the heteroduplex is detected with specific antibodies (Hybrid Capture-Digene). According to another method, the Southern method is used for the detection and genotyping of HPV genotypes. These methods have the disadvantage of being relatively insensitive and partially non-specific. In the case of Hybrid Capture, many publications describe various cross-reactions leading to false positives in a clinical setting. The authors reported that cross-reactions were only acceptable with high DNA cut-off control (1 ng / ml), highlighting the undesirable link between sensitivity and specificity.

This problem does not exist with amplification methods, as the reaction responsible for the sensitivity (amplification) is carried out separately.

Generally, the amplification techniques differ with regard to the selected amplified genome segment, the number of primers, and the detection technique used. The most commonly used starters are

GP5 + - GP6 +, MY9-MY11 and various type specific PCR reactions.

The most commonly used detection techniques are sequence specific hybridization, restriction fragment length polymorphism (RFLP) and line probe assay (LiPA). In addition to these assays, the sequencing of amplicons and degradation of thymidine by dUTP incorporation are also used, but less frequently.

The analytical characteristics of the amplification techniques vary widely. The methods can be characterized by the amplified genotypes, the analytical sensitivity of the genotype amplification, and the specificity and reliability of detection. In this regard, an array of degenerate primers

MY9-MY11 is considered to be the reference reaction. In the case of the MY9-MY11 system, there is a LiPA hybridization detection system (Innogenetics). The major disadvantage of the MY9-MY11 system is that it is difficult to control the synthesis of degenerate primers, so that the relative ratio of primer types produced in the synthesis varies from synthesis to synthesis, which can cause unpredictable changes in the analytical course of the PCR reaction; second, this reaction can amplify the smallest number of types compared to other widely used reactions. It is well known from the literature that the system can only amplify the 51 genotype if primers specific for the HPV 51 genotype are added to the reaction. When using degenerate primer synthesis, the relative ratio of primer types cannot be changed and it is not possible to adjust the primer ratio to achieve better analytical performance and a balanced amplification of genotypes.

The GP5 + - GP6 + response solves this problem only by using two carefully selected pairs of primers - optimized for HPV sex sequences - the dual primer systems are easy to handle, but the system flexibility is lower. The GP5 + - GP6 + system can amplify many known HPV genotypes, but the analytical features of the system are not optimal (sensitivity is not balanced for different genotypes) and the dual primer approach is limited in terms of optimization, e.g. the balance of detection sensitivity for individual genotypes is very problematic (except for limited optimization of the melting point and MgCl2 concentration). It is difficult to adapt the GP5 + - GP6 + system to the amplification of other genotypes, which will in any case have an impact on its future use, as the need to detect new genotypes is constantly emerging. Genotype identification is not correctly resolved.

Another widely known genotype-specific amplification method is the L1C method: two-starter design, in two versions, one uses (with the LC1 primer) the L1C2 primer or the new L1C2 to amplify further genotypes. A detailed description of the L1C amplicon can be found in the literature [Jpn. J. Cancer Research 82,524-531 (1991)].

In principle, two criteria must be met for detection by post-amplification methods: applicability in routine diagnostics (ease, cost, time) and the requirement for an appropriate level of discrimination. A significant number of methods are not appropriate with regard to discrimination. Thus, the use of RFLP is limited since the number of diagnostic restriction sites is not sufficient due to the short amplified regions, and therefore often genotypes can only be classified into groups. In another example, in an SSCP technique it is difficult to relate complex SSCP patterns to genotypes, and furthermore, the certainty of this reaction is not satisfactory.

The ability to discriminate is particularly important from a diagnostic point of view to meet the requirements of legislators. In terms of simplicity and discrimination, sequencing is ideal as it has been automated and can thus detect multiple genotypes (or even subtypes of them) if the sample is not a mixture of genotypes. However, in practice it is not widely used because it is expensive and time consuming, its use in routine diagnostic laboratories is unacceptable, and in mixed samples none of the genotypes can be determined.

The advantage of hybridization methods is that their ability to discriminate or the stringency of conditions can be easily changed, as several reaction parameters can vary widely, and some solutions can be easily automated, the reaction is less costly, and in the case of parallel introduction (for some forms) even the required time is not required. significant.

Thus, there is a need for a new HPV amplification / detection method that overcomes the drawbacks of the current methods and is cheap, easy to replicate and automate. The invention enables an amplification and hybridization assay to be carried out in which the primers are independently synthesized molecules such that their relative ratio can be easily controlled and optimized, and the amplification has a balanced sensitivity. Hybridization reactions are conducted largely in a similar manner satisfying the criteria of a cheap, fast, flexible and automated reaction.

An improved method for the detection and genotyping of human papillomavirus (HPV) has now been developed. HPV genomic regions have been defined that are suitable for the design of HPV genus specific and HPV genotype specific oligonucleotide probes and are conveniently located in close proximity and within a single amplicon. The sequences of genus specific and genotype specific probes are disclosed. An optimized reagent preparation kit and method was also developed suitable for the amplification and detection of an extended set of HPV genotypes ("kit").

PL 209 415 B1

The invention relates to an amplification primer mixture comprising primers L1C1, L1C2, new L1C2 of SEQ ID NO: 73-75, SEQ ID NO: 37-40 and SEQ ID NO: 35, and optionally at least one primer selected from the group consisting of SEQ ID primers. NO: 70-72 and SEQ ID NOS: 1-34 and 36.

The invention also relates to the use of the primer mixture as defined above for the amplification of genotypes 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 16, 18, 20, 24, 26, 28, 29, 30, 31, 32. , 33, 34, 35, 36, 37, 39, 40, 41, 42, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 66, 67, 68, 72 , 74 and 77 of the human papillomavirus.

The invention also relates to a method for detecting one or more HPV genotypes in biological samples, comprising the steps of:

a) preparing one or more amplicons with the primer mixture as defined in claim 1; 1, from nucleic acid molecules extracted from a biological sample; and b1) hybridizing the amplicon obtained in (a) under stringent conditions with a hybridization probe specific for the genotype type; and optionally b2) hybridizes the amplicon obtained in (a) under stringent conditions with an HPV genus specific probe.

Preferably, the HPV genus specific probe is:

i) HPV consensus probe or primer hybridizing to SEQ ID NO: 76, 78, 80,

82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 110;

ii) a probe complementary to the probe in (i); or iii) a sequence selected from SEQ ID NOS: 41-49.

Preferably, the genotype specific hybridization probe is:

i) a probe specific for the HPV genotype hybridizing with the sequence of SEQ ID NO: 77, 79, 81, 83,

85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109 or 111;

ii) a probe complementary to the probe in (i); or iii) a sequence selected from SEQ ID NOS: 50-57.

Preferably, the method detects low and high risk HPV genotype groups. Preferably, the method further comprises detecting the synthetic oligonucleotide as an internal template.

Preferably, the sequence of SEQ ID NO: 68 is used as the internal template and SEQ ID NO: 69 is used as a hybridization probe for its detection.

The invention further relates to a HPV detection and typing reagent kit comprising:

i) a primer mixture as defined in claim 1 1;

ii) optionally an oligonucleotide as internal standard, preferably of SEQ ID NO: 68;

iii) hybridization probes containing or hybridizing to SEQ ID NO: 77, 79, 81,

83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109 or 111 and possibly with the sequence of SEQ ID NO: 76, 78, 80, 82, 84, 86, 88 , 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 110; and iv) optionally hybridization probes, preferably of SEQ ID NO: 69, detecting the internal standard.

Disclosed herein are human papillomavirus genomic regions that are within a consensus amplicon (i.e., a region that can be amplified using primers that bind to the conserved amplicon flanking sequences), wherein these genomic regions are characterized by containing a DNA sequence specific to the amplicon. for genotype which is useful for the design of genotype specific hybridization probes.

Also disclosed herein is another HPV genomic region in this consensus amplicon, the genomic regions being characterized by containing a conserved HPV genus specific DNA sequence that is useful for designing genus specific hybridization probes.

An essential element of the invention is the presence of two genomic segments within one consensus amplicon which is suitable for designing genotype and genotype specific hybridization probes.

In general, the method of the invention can be used to amplify / detect a given group of HPV genotypes, leading to detection of HPV genomic DNA with genus specific probes and collective (group) or individual genotyping of HPV genomes. This method can be used to determine a risk or to identify those individuals at risk

The occurrence of subsequent conditions or diseases, caused by the HPV viruses or associated with the HPV viruses found in the patients, at a given time, in particular with their type-specific detection. The method of the invention is also suitable for screening a given population for the presence of HPV, and for enhancing, supporting or confirming a cytological diagnosis in a given individual (screening).

In order to facilitate a better understanding of the invention, several terms are defined below.

The terms "nucleic acid" and "oligonucleotide" refer to probes, primers and other short DNA, RNA, PNA (peptide nucleic acid) and other chemical oligomers that are capable of sequence-specific DNA, RNA or PNA binding.

The term "hybridization" refers to the sequence-specific binding of two nucleic acid sequences. The conditions used largely determine the stringency of hybridization, so that hybridization can occur under less stringent conditions, even if the nucleic acids are not exactly complementary. In some cases, it may be necessary for the nucleic acid probe to bind to a group of sequences that are more closely related or distantly related to each other. Those skilled in the art of nucleic acid technology can determine the conditions under which binding will be sufficiently specific or aspecific.

The term "probe" refers to a set of oligonucleotides that show sequence specific hybridization in the presence of complementary or partially complementary nucleic acids. The structure of the oligonucleotides can be modified to allow post-hybridization steps to take place or to change their hybridization properties.

The term "type-specific probe" refers to a set of oligonucleotides that under stringent conditions bind only to a target region that is exactly complementary to them. Suitable hybridization conditions to meet this requirement are well known (see, e.g., Sambrook et al., 1985, Molecular Cloning Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. USA). Generally, stringent conditions are selected to be about 5 ° C lower than the melting point (Tm) of the specific sequence at a defined ionic strength and pH value. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the probe is bound in the presence of a suitable complementary target molecule. Relaxing the stringency of the hybridization conditions (e.g., increasing the salt concentration or lowering the temperature) will allow for the binding of imprecise complementary sequences. In the case of an imperfectly complementary template, nucleotides that cannot bind to the template nucleic acid in the template are "mismatched nucleotides."

The term "primer" refers to nucleotides that can act as an initiation point for DNA synthesis (start) under conditions where the synthesis of a primer extension product complementary to the nucleic acid strand is induced on the nucleic acid template, i.e. in the presence of four different nucleoside triphosphates and a polymerizing agent ( i.e. DNA polymerase or reverse transcriptase) in an appropriate buffer and at the appropriate temperature. The primer is preferably a single-stranded DNA molecule. A suitable primer length is typically 15-40 nucleotides. The primer does not need to reflect the exact sequence of the template, and thus, by varying the binding (reaction) temperature, a group of similar target molecules can serve as a template for synthesis (consensus amplicon). Chemical groups with certain desirable characteristics can be used to label the primer oligonucleotide to allow it to bind to the solid phase or for other purposes.

The term "primer" as used herein also refers to a group of sequentially related oligonucleotides, wherein the group of oligonucleotides is capable of initiating synthesis (as described above) on a group of template sequences. In addition, members of the group can include oligonucleotides that can mismatch with some or all of the members of a given set of template nucleic acids. However, under appropriate conditions, these primers may also participate in the initiation of the synthesis. The term "consensus primers" refers to a primer or group of primers that can be used to initiate the synthesis of certain regions of related template nucleic acids. A characteristic feature of these regions is that their variability is significantly lower than that of the total nucleic acid, i.e. they are conserved, and therefore on these sequences selected consensus primers can initiate synthesis for an entire group of nucleic acid template sequences. The consensus primer need not be a single primer, it may be a primer group.

The term "thermostable polymerase enzyme" refers to an enzyme that is relatively stable at 95 ° C and catalyzes the polymerization of nucleoside triphosphates to produce primer extension products that are complementary to one of the nucleic acid strands of a target sequence. Cleanse 6

A combined thermostable polymerase enzyme is described in US Patent No. 4,889,818, which is hereby incorporated by reference and is commercially available from, for example, Applera.

In accordance with the invention, the amplification of DNA is carried out by the polymerase chain reaction (PCR) disclosed in U.S. Patent Nos. 4,683,195, 4,683,202 and 4,965,188.

According to the invention, optimized PCR conditions were developed to amplify more different HPV genotypes with optimization of primer concentrations, primer sequences and temperature cycling conditions. In the first part of the amplification, amplification takes place with ever-increasing stringency, in order that amplification of genotypes whose primers contain more nucleotide mismatches may start to occur with the same efficiency as other genotypes, but then increasing the binding temperature shifts the reaction towards using those primers which are present in greater amounts. With an optimized primer mix, this process will theoretically shift primer binding to the sequence towards the consensus sequence, thus making it possible to amplify genotypes with balanced sensitivity.

While the polymerase chain reaction is the preferred amplification method, the genomic regions and oligonucleotides mentioned can be used in any known method. For example, the ligase chain reaction (Wu and Wallace 1989, Genomics 4: 560-569), the TAS amplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86: 1173-1177) and self-sustained sequence replication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1874-1878) may also be suitable for the correct amplification of the target sequence. Similarly, in the Q-e-replicase system (Kramer and Lizardi, 1989, Nature 339: 401-402) sequence specific probes can be amplified.

According to the invention, primers are selected from the group of more or less complementary sequences suitable for amplifying HPV consensus amplicons. Efficient initiation of synthesis is achieved in the presence of a mismatch nucleotide using carefully selected hybridization temperatures and relative primer concentrations.

In a preferred embodiment of the invention, the primers of the invention (SEQ ID NOS: 1-40, 70-72) are used with known primers (SEQ ID NOS: 73-75) in the form of suitable reagents. This enables an extended set of HPV genotypes to be detected, of at least 47 known genotypes. Example 4 shows that the amplification of the HPV-35 genotype according to the invention can be performed by including a primer with SEQ ID NO: 37 in the reaction. Without this primer and in the presence of only known primers in the reaction, the HPV-35 genotype is not amplified. Another embodiment of the invention relates to type-specific primers for the HPV L1 gene that comprise the nucleotide sequences described in SEQ ID NOs: 1-36.

In another embodiment, the invention relates to a primer mixture comprising L1C1, L1C2, or novel L1C2 primers and primers selected from SEQ ID NOS: 1-40, 70-72. Moreover, the invention relates to the use of such a primer mixture for the amplification of the HPV 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 16, 18, 20, genotypes.

24, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 40, 41, 42, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 66, 67, 68, 72, 74 or 77.

Amplicons are produced by amplification using the aforementioned primer mix according to the invention. An essential element of the invention is the presence of genus and type specific genomic segments in the amplicons. These genomic segments allow for highly specific hybridization and detection. They are characterized by a genotype-specific variable extension of the genomic segment at the 3'-end of the amplicon (in the 3'-5 'direction) from -80 bp to -30 bp. These genomic segments, approximately 40 bp in length, are double-stranded DNA sequences shown in SEQ ID NOs: 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 , 107, 109, 111.

Detection also enables the presence of another genomic segment of amplicons, extending from the 3'-end of the amplicon (in the 3'-5 'direction) from -150 bp to -105 bp and characterized by low complexity, highly conserved sequences between genotypes that show specificity for type of HPV. These genomic segments are double-stranded DNA, typically 23 bp, and their upper strand has the following sequences: SEQ ID NO: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110.

In the hybridization detection of amplicons, generic or type-specific probes designed for the above-mentioned genomic segments are used. Generic oligonucleotide probes are generally applicable to the detection of HPV amplicon, i.e. amplified HPV DNA, while type-specific probes can be used for further typing, i.e.

Individual genotypes are detected. Both hybridization probes can be DNA, RNA or PNA in nature.

The generic probes used in the method of the invention have similar properties to consensus primers, except that the 5 'end is not preferred as the position of a mismatched pair of nucleotides. In the invention, they are used as probes, and these generic probes can be used both as hybridization probes and primers.

Generic (consensus) hybridization probes or primers are oligonucleotides that contain one of the sequences listed in SEQ ID NOS: 41-49

In a preferred method of the invention, the following probes are used as generic probes: SEQ ID NOS: 41-49.

In the case of type-specific probes, the probes are 100% complementary to the sequence of the corresponding genotype. When designing them, it is important to exclude the possibility that other genotypes show a high degree of complementarity with the probe or part of it. Since the type-specific segment is approximately 40 bp in length in the amplicons of the invention, it is possible to select even overlapping probe sequences that are most relevant both theoretically and experimentally.

Example 5 shows that it is possible to design and use suitable probes that have high specificity (compared to the 70 genotypes tested) and retain their specificity even when using hybridization at room temperature conditions. Since probe hybridization is suitably specific under identical hybridization conditions, a mixture of probes can be used. Thus, from a practical point of view, more uniform reaction conditions can be used. Thus, in order to practice the invention, there are disclosed type-specific probe sequences that can be used in HPV amplification and detection and genotyping assays, which include the sequences given in SEQ ID NOS: 50-67.

While the invention relates to any form of hybridization, solid phase hybridization is preferred under industrial conditions.

In so-called solid-phase (forward) hybridization, the probes bind to the immobilized target nucleic acid (amplicon), while in reverse hybridization, the target nucleic acid (amplicon) binds to the immobilized probes. In this process, unbound reaction products are removed with various washing solutions. In the former case, the probe must be properly labeled for further development, while the amplicon is labeled inversely. Both methods can be carried out in microtiter plates. Since the immobilization capacity is limited, the forward hybridization method is preferred in the present invention due to the large number of individual probes being used to form the probe mixture.

The method described in US Patent No. 6,214,979 in which probes are added to the reaction during amplification may also be useful. During the process, the activity of the 5'-3 'egozene-Taq glutase polymerase degrades the probes and detection of the degradation products produced can be used to detect the presence of the target nucleic acid. However, other, in most cases hybridization based, so-called real-time detection systems are also known, but these differ only in the manner of implementation and detection, but not theoretically in a different embodiment of the sequence-specific probe-amplicon binding.

The amplicons may be labeled for immobilization. It is advantageous in the present invention to label the primers with biotin and to use the labeled primer in the amplification reactions. In the presence of solid phase adsorbed avidin or streptavidin, biotin causes amplicon immobilization as a result of highly specific and very stable avidin-biotin binding. In a given reaction, only primers hybridizing to one or the other strand are biotinylated so that the other strand can be removed prior to hybridization.

For detection purposes, the probes may be labeled, where the labels may be detected by various methods, e.g. detection methods based on fluorescence, radioactivity, colorimetry, X-ray diffraction, absorption, magnetism, enzymatic activity, etc. Thus, appropriate labeling may include, but is not limited to the following: fluorophores, chromophores, isotopes, electron-dense substances, enzymes or ligands capable of forming specific binding. Combinations of these above-mentioned methods can also be used.

Specific binding of fluorescein to the anti-fluorescein HRPO antibody, which is detected by the oxidation reaction by horseradish peroxidase in the presence of a substrate, can be used.

HPPA and H2O2 (see example 2). Biotinylated forward or reverse primers can also be used. Both labeled probes and anti-fluorescein-HRPO antibody are commercially available. Chemical reagents required for washing and various solutions are also generally available. A system that employs alkaline phosphatase or any other enzyme whose activity can be detected can also be prepared or used. In addition, luminometry with fluorescence detection or colorimetry are also acceptable detection methods for use in medical diagnostics.

The inclusion of an internal standard during the diagnostic application of the method according to the invention makes it possible to recognize false negative reactions and is advantageous from a diagnostic point of view. Various DNAs of artificial and natural origin can be used as an internal template.

Those systems that do not increase the number of primers used in the reaction are preferred, and the amount and analytical properties of target nucleic acid as internal standard are suitably standardized. According to the invention, an artificial nucleic acid sequence (SEQ ID NO: 68) is added to the sample in the form of a recombinant plasmid (example 6).

According to the invention, the detection of the internal standard is carried out in parallel with the hybridization detection of the HPV DNA and only the development of the substrates is separated. The probe is labeled with digitoxigenin (SEQ ID NO: 69) and detected with an alkaline phosphatase-conjugated anti-digitoxigenin antibody.

However, other detection methods are also suitable for detecting the internal probe.

However, detection of the internal standard can also be performed on the basis of differences in mobility using agarose gel electrophoresis or other suitable methods.

For the amplification and detection of HPV DNA, the method according to the invention is suitable for obtaining a harmonized unit of reagents (kit). In this embodiment, the kit may contain all or any combination of the following reagents and other reagents: primers, primer mix, buffers, thermostable polymerase, HPV DNA as positive control, non-HPV DNA, DNA as internal standard, probes or probe mix, antibody conjugate -enzyme.

The description of the primer and probe sequences of the invention is exemplary only. One skilled in the art can design many generic and type-specific probe variants using either the generic or the type-specific HPV genomic region, and thus variants are within the scope of the invention, provided that they are selected from the genomic region disclosed herein.

The invention is illustrated in more detail in the following Examples illustrating the invention. Although the method on which the invention is based is useful for the amplification of any HPV genome, only the HPV sex genomes, which are of greater medical importance, are used in the following examples.

Example 1. Synthesis of oligonucleotide primers

The oligonucleotide primers used in the method of the invention were obtained from a commercial source (IDT, USA). The primers were synthesized with a 5'-amino group. NHS-ester was used for biotin, fluorescein and digitoxigenin labeling. The labeled nucleotides were purified by HPLC.

Example 2. Sample processing, DNA preparation

Samples taken by gynecologists with the Cytobrush were transported in PBS (10 mM phosphate buffered saline, pH = 7.4, Sigma, NaCl 138 mM, KCl 2.7 mM). The pretreatment of the samples was done in the test tubes: before lysis, the samples were centrifuged (2000 g, 10 minutes), the supernatants were discarded and 1 ml of PBS solution was added, vortexed and again centrifuged discarding the supernatrant. Finally, 250 μΐ of lysis solution (0.5 mg / ml Proteinase-K, 0.01 M TRIS-HCl pH = 8, 0.001 M EDTA pH = 8, in distilled water) was added to the samples, and this solution contained the standard internal for HPV assay (SEQ ID NO: 68), the solution was vortexed and incubated for 30 minutes at 56 ° C. From that moment on, all operations on the samples in the liquid state were performed in the TECAN RSP150 robot: 200 μl of the binding solution (5.5 M GUSCN, 20 mM EDTA, 10 mM TRIS-HCl pH = 6.5, 65 mM dithiothreitol, g / g / 1 silica, SIGMA catalog no: 28.851-3, distilled water) and silica were separated by vacuum filtration from the soluble components.

Filtration was performed again to wash the silica twice with 200 μl of the silica-free binding solution (5.5 M GUSCN, 20 mM EDTA, 10 mM TRIS-HCl pH = 6.5, 65 mM dithiothreitol, distilled water) and 200 μl of the washing solution ( 25% isopropyl alcohol, 25% ethanol 96%, 50% distilled water, 0.1M NaCl), 200 μl 96% ethanol was finally used.

PL 209 415 B1

After air drying the silica, the DNA was eluted with 200 µl of a 10 mM TRIS solution, pH = 8.0. The eluted DNA was stored frozen at -20 ° C until further use.

Example 3. General description of HPV detection

Amplification

The total volume of the reaction mixture was 25 µl with the following components: 10 µl of DNA, 2.5 µl of 10 x polymerase buffer (final concentration: 10 mM TRIS-HCl (pH = 9.0), 50 mM KCl, 0 , 1% Triton X-100 (Promega)), 2 mM MgCl ₂ , 250 pM each dNTP (ATP, CTP, GTP, TTP), 4 pM primer mix: SEQ ID NO: 35, 37-40, 73-75 and 1U Taq DNA polymerase (Promega). The reaction was performed in a GeneAmp 9700 PCR thermocycler with the following parameters: cycle 1: 4 minutes at 95 ° C; cycles 2-40: 30 s at 94 ° C, 1 minute at 48 ° C and 45 s at 72 ° C; cycle 41: 3 minutes at 72 ° C.

Hybridization and detection

Hybridization was performed on a solid phase. 24 hours earlier, black, 96-well polystyrene plates (Costar) were coated with streptavidin (0.02 mg / ml streptavidin in PBS solution). The plates were incubated at room temperature and 24 hours later the plates were washed with 250 µl of washing solution [25 mM TRIS pH = 7.5, 125 mM NaCl, 20 mM MgCl 2, 3% Tween-20]. 20 µl of the PCR reaction product was diluted with 140 µl of distilled water and 5 µl of this solution was mixed with 45 µl of binding buffer [25 mM TRIS pH = 7.5, 125 mM NaCl, 5 mM EDTA-Na2, 5 x Denhardt's solution, 0.1 % Tween-20] and dispensed into the wells of a streptavidin coated plate.

The reaction mixture was incubated at room temperature for 30 minutes with constant agitation. Then 50 µl of elution buffer [100 mM NaOH, 300 mM NaCl] was added to the mixture, incubated for 3 minutes at room temperature and the plates were washed three times with 250 µl of washing solution [25 mM TRIS pH = 7.5, 125 mM NaCl, 20 mM MgCl2 , 3% Tween-20]. After washing, 50 µl of hybridization buffer (5 × SSC (0.3M Na citrate, pH = 7, 3M NaCl), 1 × Denhardt's solution, 0.1% SDS] containing fluorescein-labeled probes (5 nM on probe).

The mixture was incubated for 30 minutes at 50 ° C with constant agitation and washed six times with 250 µl of high stringency washing solution [0.05 x SSC, 0.3% Tween-20]. Then, 50 µl of coupling buffer [25 mM TRIS pH = 7.5, 125 mM NaCl, 2 mM MgCl2, 0.3% Tween-20, 1% BSA] containing anti-Fluorescence-POD antibody (Roche) was added (0 .0015 E / reaction).

The plates were incubated for 30 minutes at room temperature with constant shaking and washed six times with 250 µl of high-stringency wash solution [25 mM TRIS pH = 7.5, 125 mM NaCl, 20 mM MgCl2, 3% Tween-20]. For development, 135 µl of substrate solution was added (5 volumes [45 mM hydroxyphenylpropionic acid (HPPA), dissolved in 0.1M TRIS-HCl buffer pH = 9.0] + 1 volume [0.6 g / liter H2O2 in 20 mM buffer Citrate Phosphate]).

To stop the reaction after 20 minutes, 65 µl of a stop solution [0.75M glycine, pH = 10.3] was added to the reaction mixture. The fluorescence signal was measured with a SpectraMax plate fluorimeter at 324/410 nm. Samples were considered positive when their value was greater than three times the mean value of three parallel negative control samples.

Example 4. Comparative amplification study

10 ng / reaction of plasmids containing the cloned HPV L1 region from different genotypes were added to the samples and used to amplify and detect HPV DNA according to the method of the invention. PCR and hybridization were performed as described in Example 3, except that the primer composition was changed. The reaction without L1F2 (SEQ ID NO: 37) did not amplify the HPV 35 genotype, while the L1F2 primer provided effective detection of the HPV 35 genotype.

Example 5. Detection of several HPV genotypes

10 ng / reaction of plasmids containing the cloned HPV L1 region from different genotypes were added to the samples and used to amplify and detect HPV DNA according to the method of the invention. The PCR reaction and hybridization were performed as described in example 3.

Attempts were made to amplify and type the following HPV genotypes: 1-24, 26-42, 45, 47-68, 72-74, 76-77, 86. Amplification of the following genotypes was demonstrated by agarose gel electrophoresis: 3, 6-7, 10 -11, 13-14, 16, 18, 20, 24, 26, 29, 30-36, 39-40, 42, 45, 51, 52-55, 58-62, 6668, 72. Using hybridization probe mix genotype specific SEQ ID NO: 41-49 (under the conditions described in example 3) the following genotypes could be detected: 3-4, 6-7, 9-14, 16, 18, 20, 24, 26, 29, 30- 37, 39-42, 45, 51, 52-55, 58-62, 66-68, 72, 74, 77.

The data show that some genotypes can only be detected by hybridization, which is not surprising as hybridization is about 10-100 times more sensitive than agaric gel electrophoresis10

PL 209 415 B1. These data also show that the genus specific hybridization allows the detection of all genotypes positive by agarose gel electrophoresis, indicating its indeed genus specific nature.

Example 6. Detection of the HPV-35 type

10 ng / reaction of plasmids containing the cloned HPV L1 region from different genotypes were added to the samples and used to amplify and detect HPV DNA by the method of the invention. PCR and hybridization were performed as described in Example 3. The probe to be hybridized was an oligonucleotide designed for the HPV 35 genotype (SEQ ID NO: 58). Attempts were made to detect the following amplicons: 3-4, 6-7, 9-14, 16, 18, 20, 24, 26, 29, 30-37, 39-42, 45, 51, 52-55, 58-62, 66-68, 72, 74, 77. The probe detected only the amplicon corresponding to the HPV 35 genotype.

Example 7. Detection of HPV with an internal standard

Detection for samples prepared according to example 2 was performed according to example 3, except that the digitoxigenin-labeled internal standard probe (the same amount as the type-specific probes) was used with the type-specific probes and antibodies were present in the conjugation step. against fluorescein-POD and against alp-ALP [1: 2000, Jackson Immuno Research].

After induction of the POD reaction (see example 2), ALP induction was performed as follows: The substrate solution contained 6.25 mg / 100 ml of 4-methyl umbeliferyl phosphate in 100 mM buffer and TRIS pH = 9.0, 0.5 mM MgCl2.

After HPPA development, the microtiter plate was washed once [25 mM TRIS pH = 7.5,

125 mM NaCl, 20 mM MgCl ₂ , 3% Tween-20] and 150 µL of substrate solution was added to each reaction well.

After 30 minutes of incubation, the fluorescence signal was measured with a SpectraMax plate fluorimeter at 355/460 nm. Samples were considered positive when their value was greater than three times the mean value of three parallel negative control samples.

The type-specific probes detected only the correct types. Detection of the Internal Control was positive with every reaction, except for those reactions where there was a strong competition between HPV amplification and the internal control. There were no inhibited reactions (HPV DNA or Internal Control DNA was amplified in all samples). The internal control adequately ruled out the possibility of a false negative.

PL 209 415 B1

<210> 4 <211> 21 <212> DNA <213> Dummy <220>

<223> PCR primer (L.1CR11)

PL 209 415 B1

PL 209 415 B1

<211> <212> <213> 21 GOUT Artificial sequence

<220> <223> PCR primer (L1CR16) <400> 10

caatacaggg tatttagaat a

<210> <211> <212> <213> 11 25 GOUT Artificial sequence

<220> <223> PCR primer (L1CF1B) <400> 11

cgtaaacgtg ttccctattt ttttg 25

<210> <211> <212> <213> 12 21 GOUT Artificial sequence

<220> <223> PCR primer (L1CR18) <400> 12

caatatagag tatttagggt g 21

<210> <211> 13 24 <212> <213> GOUT Artificial sequence

<220> <223> PCR primer (L1CF31) <400> 13

cgtaaacgtg tatcatattt tttt 24

<210> <211> <212> <213> 14 21 GOUT Artificial sequence

<220> <223> PCR primer (L1CR31) <400> 14

caatataggg tatttagggt t 21 <210> 15

<211> 25 <212> GOUT <213> Artificial sequence

<220> <223> PCR primer (L1CF33)

PL 209 415 B1

PL 209 415 B1

<210> 26 <211> 21 <212> DNA

PL 209 415 B1

PL 209 415 B1

PL 209 415 B1

<210> 42 <211> 23

<212> DNA <213> Artificial sequence <220>

<223>

genus specific hybridization probe (PK2) <400> 42 cgcacaagca tctattatta tgc <210> 43 <211> 23 <212> DNA <213> Dummy <220>

<223>

genus specific hybridization probe (PK3) <400> 43 cgcacaagca tattttatca tgc <210> 44 <211> 23 <212> DNA <213> Artificial sequence <220>

<223> genus specific hybridization probe (PK4) <400> 44 cgcaccagta tattttatca tgc <210> 45 <211> 23 <212> DNA <213> Artificial sequence <220>

<223>

genus specific hybridization probe (PK5) <400> 45 cgcacaagca tttactatca tgc <210> 46 <211> 23 <212> DNA <213> Artificial sequence <220>

<223>

genus specific hybridization probe (PK6) <400> 46 cgcaccaact acttttacca tgc

<210> 47 <211> 23 <212> GOUT <213> Artificial sequence <220>

PL 209 415 B1 <223> genus specific hybridization probe (PK7) <400> 47 cgtaccagta ttttctacca cgc <210> 48 <211> 23 <212> DNA <213> Artificial sequence <220>

<223> genus specific hybridization probe (PK8) <400> 48 cgcacaggca tatattacta tgc <210> 49 <211> 23 <212> DNA <213> Artificial sequence <220>

<223>

genus specific hybridization probe (PK9) <400> 49 cgcaccaaca tatattatca tgc <210> 50 <211> 20 <212> DNA <213> Artificial sequence <220>

<223> HPV-S specific hybridization probe (P6) <400> 50 ttttgttagc ccgttttatg <210> 51 <211> 23 <212> DNA <213> Artificial sequence <220>

<223>

HPV-11 specific hybridization probe (Pil) <400> 51 acaactgttt ttgttaactt ttt <210> 52 <211> 20 <212> DNA <213> Artificial sequence <220>

<223>

hybridization probe specific for HPV-42 (P42) <400> 52 tgtcttattt ggcctttttg

PL 209 415 B1 <210> 53 <211> 20 <212> DNA <213> Artificial sequence <220>

<223> hybridization probe specific respect HPV-44 (P44) <400> 53 gtcttgtttg ctggtcgtat twenty <210> 54 <211> 25 <212> □ NA <213> Artificial sequence <220> <223> hybridization probe specific respect HPV-1S (P15) <400> 54 ctaatatttt gttattgtta ggttt 25 <210> 55 <211> 19 <212> GOUT <213> Artificial sequence <220> <223> hybridization probe specific respect HPV-18 (P18) <400> 55 tatcctgctt attgccacc 19 <210> 55 <211> 22 <212> GOUT <213> Artificial sequence <220> <223> hybridization probe specific respect HPV-31 (P31) <400> 55 ggattgtcag atttaggtat gg 22 <210> 57 <211> 23 <212> GOUT <213> Artificial sequence <220> <223> hybridization probe specific respect HPV-33 (P33)

<400> 57 tttttagcgt tagtaggatt ttt 23.-9 ι η .. R fi

PL 209 415 B1 <211> 25 <212> DNA <213> Artificial sequence <220>

<223> hybridization probe specific respect HPV-35 (P35) <400> 58 tgctatttta ttagaatctt gtttt 25 <210> 59 <211> 19 <212> GOUT <213> Artificial sequence <220> <223> hybridization probe specific respect HPV-39 (P39) <400> 59 accaccattc atacccact 19 <210> 60 <211> twenty <212> GOUT <213> Artificial sequence <220> <223> hybridization probe specific respect HPV-45 (P45) <400> 60 acctgcacca ttaggtacaa twenty <210> 61 <211> twenty <212> GOUT <213> Artificial sequence <220> <223> hybridization probe specific respect HPV-51 (P51) <400> 61 gcgttgaggt tttaggtatt twenty <210> 62 <211> 21 <212> GOUT <213> Artificial sequence <220> <223> hybridization probe specific respect HPV-52 (P52)

<400> 62 caattaccac tactggtgtt t 21 <210> 63 <211> 21 <212> DNA <213> Dummy sequence

PL 209 415 B1 <220>

<223> hybridization probe specific for HPV-56 (P56) <400> 63 tgtttgtttt ggtattgtcc t <210> 64 <211> 20 <212> DNA <213> Artificial sequence <220>

<223> HPV-58 specific hybridization probe (ΡΞ8) <400> 64 gttattggga cttttgatgg <210> 65 <211> 20 <212> DNA <213> Artificial sequence <220>

<223> HPV-59 (P59) specific hybridization probe <400> 65 tcctgtctac cattaccacc <210> 66 <211> 20 <212> DNA <213> Artificial sequence <220>

<223> HPV-66 specific hybridization probe (P66) <400> 66 ttggtaccag atttggaaac <210> 67 <211> 20 <21?> DNA <213> Artificial sequence <220>

<223> HPV-68 specific hybridization probe (P68) <400> 67 cccagacata ggaaccttaa <210> 68 <211> 198 <212> DNA <213> Artificial sequence <220>

<223> Internal control oligonucleotide molecule <400> 68

PL 209 415 B1

cgtaaacgtt ttccctattt ttttagtcaa tgagacgggt aatgacgata cagtatgacg 60 atagagtaga tagatagaga tagataccca tatacagata atgacataga tccccataga 120 cagtttatac agatcagtag cagtttttat atatgagatg atgataggac acaccagcaa 180 tatagggtat ttagggta 198

<210> 69 <211> 21 <212> DNA <213> Dummy <220>

<223> Amplicon specific internal control hybridization probe (IC-P1) <400> 69 tgacatagat ccccatagac a <210> 70 <211> 24 <212> DNA

213> Dummy <220>

<223> PCR primer (L1F5) <400> 70 cgtaaacgta ttccctattt tttt <210> 71 <211> 24 <212> DNA <213> Dummy <220>

<223> PCR primer (L1F6) <400> 71 cgtaaacgtt ttccatattt tttt <210> 72 <211> 21 <212> DNA <213> Dummy <220>

<223> PCR primer (L1R3) <400> 72 cagtacagag tttttagagt g <210> 73 <211> 24 <212> DNA <213> Dummy sequence

PL 209 415 B1 <220>

<223> PCR primer (LICI) <400> 73 cgtaaacgtt ttccctattt tttt <210> 74 <211> 21 <212> DNA <213> Dummy <220>

<223> PCR primer (L1C2) <400> 74 caatacagag tatttagggt a 21 <210> 75 <211> 21 <212> DNA <213> Dummy <220>

<223> PCR primer (new L1C2) <400> 75 caatataggg tatttagggt a <210> 76 <211> 23 <212> DNA <213> Human papillomavirus type 6 <220>

<221> Miscellaneous <223> Probe binding site of type-specific HPV-6 amplicon <400> 76 cgcaccaaca tattttatca tgc <210> 77 <211> 39 <212> DNA <213> Human Type 6 Papillomavirus <220>

<221> various features <223> Type-specific HPV-6 amplicon probe binding site <400> 77 ccttattttt ccataaaacg ggctaacaaa actgttgtg <2 10> <211> <212> <213>

GOUT

Human papillomavirus type 11 <220>

PL 209 415 B1 <221> various features <223> Genus specific HPV-11 amplicon probe binding site <400> 78 cgcaccaaca tattttatca tgc <210> 79 <211> 39 <212> DNA <213> Human papillomavirus type 11 < 220>

<221> various characteristics <223> Type-specific HPV-11 amplicon probe binding site <400> 79 ccatattact ctatcaaaaa agttaacaaa acagttgta <210> 80 <211> 23 <212> DNA <213> Human papillomavirus type 42 <220>

<221> miscellaneous <223> type-specific HPV-42 amplicon probe binding site <400> 80 cgcaccaact acttttacca tgc <210> 81 <211> 42 <212> DNA <213> Human papillomavirus type 42 <220>

<221> various characteristics <223> Type-specific HPV-42 amplicon probe binding site <400> 81 ccttattact ctattacaaa aagccaaata agacatctat cc <210> 82 <211> 23 <212> DNA <213> Human papillomavirus type 44 <220>

<221> various features <223> Genus specific HPV-44 amplicon probe binding site.

<400> 82 cgcaccaaca tatattacca tgc

PL 209 415 B1 <210>

<211>

<212>

<213>

<220>

<221>

<223>

GOUT

Human papillomavirus type 44 different characteristics

Probe binding site of type-specific HPV-44 amplicon <400> 83 ccttattttg ccatacgacc agcaaacaag acacttgtg 39

<210> 84 <211> 23 <212> GOUT <213> Human papillomavirus type 16 <220> <221> different features <223> Probe binding site u-type amplicon <400> 84

cgcacaaaca tatattatca tgc specific to <210>

<211>

<212>

<213>

<220>

<221>

<223>

GOUT

Human papillomavirus type 16 different characteristics

Probe binding site of type-specific HPV-16 amplicon <400> 85 ttttcctatt aaaaaaccta acaataacaa aatattagtt <210> 86 <211> 23 <212> DNA <213> Human papillomavirus type 18 <220>

<221>

<223>

different features

Probe binding site u-genus-specific HPV-18 amplicon <400> 86 cccacaagca tattttatca tgc <210> 87 <211> 41 <212> DNA <213> Human papillomavirus type 18 <220>

<221> various features

PL 209 415 B1 <223> Probe binding site of type-specific HPV-18 amplicon <400> 87 attttagggt tcctgcaggt ggtggcaata agcaggatat t 41 <210> 88 <211> 23 <212> DNA <213> Human papillomavirus type 31 <220>

<221> various characteristics <223> Probe binding site of type-specific HPV-31 amplicon <400> 88 cgaaccaaca tatattatca cgc 23 <210> 89 <211> 40 <212> DNA <213> Human papillomavirus type 31 <220>

<221> various characteristics <223> Type-specific HPV-31 amplicon probe binding site <400> 89 ttccatacct aaatctgaca atcctaaaaa aatagttgta 40 <210> 90 <211> 23 <212> DNA <213> Human papillomavirus type 33 <220>

<221> miscellaneous <223> genus specific HPV-33 amplicon probe binding site <400> 90 cgcacaagca tttattatta tgc 23 <210> 91 <211> 40 <212> DNA <213> human papillomavirus type 33 <220>

<221> various characteristics <223> Probe binding site of type-specific HPV-33 amplicon <400> 91 ttctattaaa aatcctacta acgctaaaaa attattggta 40

PL 209 415 B1 <210>

<211>

<212>

<213>

<220>

<221>

<223>

GOUT

Human papillomavirus type 35 different characteristics

Probe binding site of genus specific HPV-35 amplicon <400> 92 cgcacaaaca tctactatca tgc 23 <210> 93 <211> 40 <212> DNA <213> Human papillomavirus type 35 <220>

<221> various features <223> Type-specific HPV-35 amplicon probe binding site <400> 93 ctatgctatt aaaaaacaag attctaataa aatagcagta

<210> 94 <211> 23 <212> GOUT <213> Human papillomavirus type 39 <220> <221> different features <223> Probe binding site of the genus amplicon <400> 94

cgcacaggca tatattatta tgc <210> 95 <211> 41 <212> DNA <213> Human Papillomavirus Type 39 <220> <221> <223>

different features

Probe binding site of type-specific HPV-39 amplicon <400> 95 attttaaagt gggtatgaat ggtggtcgca agcaggacat t <210>

<211>

<212>

DNA <213> Human papillomavirus type 45 <220>

<221> various features

PL 209 415 B1

<223> Probe binding site of HPV-45 genus amplicon specific respect <400> 96 cgcacaagca tattttatca tgc 23 <210> 97 <211> 40 <212> GOUT <213> Human papillomavirus type 45 <220> <221> different features <223> HPV-45 amplicon probe binding site specific respect type <400> 97 tagggttgta cctaatggtg caggtaataa acaggctgtt 40 <210> 98 <211> 23 <212> GOUT <213> Human papillomavirus type 51 <220> <221> different features <223> HPV-51 amplicon probe binding site specific respect kind <400> 98 cgcaccggca tatattacta tgc 23

<210> 99 <211> 40 <212> GOUT <213> Human papillomavirus type 51 <220> <221> different features <223> Type-specific HPV-51 amplicon probe binding site <400> 99 ctattttcca atacctaaaa cctcaacgcg tgctgctatt 40

<210> 100 <211> 23 <212> GOUT <213> Human papillomavirus type 52 <220> <221> different features <223> Probe binding site for HPV-52 amplicon specific for

type u <400> 100 cgcacaagca tctattatta tgc 23 <210> 101

PL 209 415 B1 <211>

<212>

DNA <213> Human papillomavirus type 52 <220>

<221> various features <223> Type-specific HPV-52 amplicon probe binding site <400> 101 taaaaacacc agtagtggta atggtaaaaa agttttagtt c 41 <210>

<211>

<212>

102

DNA <213> Human papillomavirus type 56 <220>

<221> miscellaneous characteristics <223> genus specific HPV-56 amplicon probe binding site <400> 102 cgcactagta tattttatca tgc 23 <210> 103 <211> 41 <212> DNA <213> human papillomavirus type 56 <220>

<221>

<223>

different features

Probe binding site of type specific HPV-56 amplicon <400> 103 ctattactct gtgactaagg acaataccaa aacaaacatt c <210>

<211>

<212>

<213>

104

GOUT

Human papillomavirus type 58 <220>

<221>

<223>

different features

Probe binding site of genus-specific HPV-58 amplicon <400> 104 cgcacaagca tttattatta tgc <210> 105 <211> 41 <212> DNA <213> Human papillomavirus <220>

<221>

<223>

different features

Type-specific HPV-58 amplicon probe binding site

PL 209 415 B1

<400> 105 ttccatcaaa agtcccaata acaataaaaa agtattagtt c 41 <210> <211> <212> <213> 106 23 GOUT Human papillomavirus type 59 <220> <221> <223> different features Probe binding site of HPV-59 genus amplicon specific to <400> 106 cgtaccagta ttttctacca cgc 23

<210> 107 <211> 41 <212> GOUT <213> Human papillomavirus type 59 <220> <221> different features <223> Type-specific HPV-59 amplicon probe binding site <400> 107 ttttaaagta cctaaaggtg gtaatggtag acaggatgtt c 41

<210> 108 <211> 23 <212> GOUT <213> Human papillomavirus type 66 <220> <221> different features <223> Type-specific HPV-66 amplicon probe binding site <400> 108 cgtaccagta tattttatca tgc 23

<210> 109 <211> 41 <212> GOUT <213> Human papillomavirus type 66 <220> <221> different features <223> Type-specific HPV-66 amplicon probe binding site <400> 109 ttattactct gtttccaaat ctggtaccaa aacaaacatc c 41

<210> 110 <211> 23 <212> DNA

<213> Human Papillomavirus Type 68 <220>

<221> various characteristics <223> Probe binding site of type-specific HPV-68 amplicon <400> 110 cgcactggca tgtattacta tgc 23 <210> 111 <211> 40 <212> DNA <213> Human papillomavirus type 68 <220>

<221> various characteristics <223> Type-specific HPV-68 amplicon probe binding site <400> 111 ttttaaggtt cctatgtctg ggggccgcaa gcagggcatt 40 <210> 112 <211> 244 <212> DNA <213> Human papillomavirus type 44 <220>

<221> miscellaneous characteristics <223> HPV-44 amplicon <400> 112

cgtaaacgtg tttccttgtt ttttgcagat gtggcggcct agtgaaaacc aggtatatgt 60 gcctcctccc gccccagtat ccaaagtaat acctacggat gcctatgtca aacgcaccaa 120 catatattac catgctagca gttctagact tcttgctgtg ggcaaccctt attttgccat 180 acgaccagca aacaagacac ttgtgcctaa ggtttcggga tttcaatata gggtttttaa 240 gatg 244

<210> 113 <211> 253 <212> DNA <213> Human papillomavirus type 35 <220>

<221> miscellaneous characteristics <223> HPV-35 amplicon <400> 113

cgtaaagcta tcccatattt ttttgcagat gtctctgtgg cggtctaacg aagccactgt 60 ctacctgcct ccagtgtcag tgtctaaggt tgttagcact gatgaatatg taacacgcac 120 aaacatctac tatcatgcag gcagttctag gctattagct gtgggtcacc catactatgc 180 tattaaaaaa caagattcta ataaaatagc agtacccaag gtatctggtt tgcaatacag 240

PL 209 415 B1 agtatttaga gta 253 <210> 114 <211> 253 <212> DNA <213> Human papillomavirus type 39 <220>

<221> miscellaneous characteristics <223> HPV-39 amplicon <400> 114

cgtaaacgta ttccctattt tttttcagat ggctatgtgg cggtctagtg acagcatggt 60 gtatttgcct ccaccttctg tggcgaaggt tgtcaatact gatgattatg ttacacgcac 120 aggcatatat tattatgctg gcagctctag attattaaca gtaggacatc catattttaa 180 agtgggtatg aatggtggtc gcaagcagga cattccaaag gtgtctgcat atcaatatag 240 ggtatttcgc gtg 253

<210> 115 <211> 25S <212> DNA <213> Human papillomavirus type 45 <220>

<221> miscellaneous characteristics <223> HPV-45 amplicon <400> 115

cgtaaacgta ttccctattt ttttgcagat ggctttgtgg cggcctagtg acagtacggt 60 atatcttcca ccaccttctg tggccagagt tgtcagcact gatgattatg tgtctcgcac 120 aagcatattt tatcatgcag gcagttcccg attattaact gtaggcaatc catattttag 180 ggttgtacct aatggtgcag gtaataaaca ggctgttcct aaggtatccg catatcagta 240 tagggtgttt agagta 256

<210> 116 <211> 250 <212> DNA <213> Human papillomavirus type 51 <220>

<221> miscellaneous characteristics <223> HPV-51 amplicon <400> 116

cgtaaacgta taccctattt ttttacagat ggcattgtgg cgcactaatg acagcaaggt 60 gtatttgcca cctgcacctg tgtctcgaat tgtgaataca gaagaatata tcacacgcac 120 cggcatatat tactatgcag gcagttccag actaataaca ttaggacatc cctattttcc 180 aatacctaaa acctcaacgc gtgctgctat tcctaaagta tctgcatttc aatacagggt 240 atttagggta 250

PL 209 415 B1 <210> 117 <211> 250 <212> DNA <213> Human papillomavirus type 56 <220>

<221> miscellaneous characteristics <223> HPV-56 amplicon <400> 117

cgtaaacgta ttccctattt ttttgcagat ggcgacgtgg cggcctagtg aaaataaggt 60 gtatctacct ccaacacctg tttcaaaggt tgtggcaacg gattcctatg yeahacgcac 120 tagtatattt tatcatgcag gcagttcacg attgcttgcc gtaggaoatc cctattactc 180 tgtgactaag gacaatacca aaacaaacat tcccaaagtt agtgcatatc aatatagggt 240 atttagggta 250

<210> 118 <211> 253 <212> DNA <213> Human Papillomavirus Type 59 <220>

<221> miscellaneous characteristics <223> HPV-59 amplicon <400> 118

ctgaaactgg ttccctattt ttttacagat ggctctatgg cgttctagtg acaacaaggt 60 gtatctacct ccaccttcgg tagctaaggt tgtcagcact gatgagtatg tcacccgtac 120 cagtattttc taccacgcag gcagttccag acttcttaca gttggacatc catattttaa 180 agtacctaaa ggtggtaatg gtagacagga tgttcctaag gtgtctgcat atcaatacag 240 agtatttagg gtt 253

<210> 119 <211>

<212>

250

DNA <213> Human Type 66 Papillomavirus <220>

<221> miscellaneous characteristics <223> HPV-66 amplicon <400> 119

cgtaaacgta ttccctattt ttttgcagat ggcgatgtgg cggcctagtg acaataaggt 60 gtacctacct ccaacacctg tttcaaaggt tgtggcaacg gatacatatg taaaacgtac 120 cagtatattt tatcatgcag gtagctctag gttgcttgct gttggccatc cttattactc 180 tgtttccaaa tctggtacca aaacaaacat ccctaaagtt agtgcatatc agtatagagt 240 gtttagggta 250

PL 209 415 B1 <210> 120 <211> 253 <212> DNA <213> Human papillomavirus type 68 <220>

<221> miscellaneous characteristics <223> HPV-68 amplicon <400> 120

cgtaaacacc ttccttattt ttttacagat ggcattgtgg cgagctagcg acaacatggt 60 gtatttgcct cccccctcag tggcgaaggt tgtcaataca gatgattatg tgacacgcac 120 tggcatgtat tactatgctg gtacatctag gttattaact gtaggccatc catattttaa 180 ggttcctatg tctgggggcc gcaagcaggg cattcctaag gtgtctgcat atcaatacag 240 agtgtttagg gtt 253

Patent claims

Claims (9)

Patent claims CLAIMS 1.A amplification primer mixture comprising primers L1C1, L1C2, a new L1C2 of SEQ ID NO: 73-75, SEQ ID NO: 37-40 and SEQ ID NO: 35, and optionally at least one primer selected from the group comprising primers of SEQ ID NOS: 70-72 and SEQ ID NOS: 1-34 and 36. 2. The use of a primer mixture as defined in claim 1 1 for the amplification of genotypes 3, 4, 6, 7, 9, 10, 11, 12, 13, 14, 16, 18, 20, 24, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 40, 41, 42, 44, 45, 51, 52, 53, 54, 55, 56, 58, 59, 60, 61, 66, 67, 68, 72, 74 and 77 human papillomavirus . 3. A method for detecting one or more HPV genotypes in a biological sample comprising the steps of: a) preparing one or more amplicons with the primer mixture as defined in claim 1; 1, from nucleic acid molecules extracted from a biological sample; and b1) hybridizing the amplicon obtained in (a) under stringent conditions with a hybridization probe specific for the genotype type; and optionally b2) hybridizes the amplicon obtained in (a) under stringent conditions with an HPV genus specific probe. 4. The method according to p. The method of claim 3, wherein the HPV type-specific probe is: i) HPV consensus probe or primer hybridizing to SEQ ID NO: 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 110 ; ii) a probe complementary to the probe in (i); or iii) a sequence selected from SEQ ID NOS: 41-49. 5. The method according to p. The method of claim 3, wherein the genotype specific hybridization probe is: i) an HPV genotype specific probe hybridizing to SEQ ID NO: 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109 or 111; ii) a probe complementary to the probe in (i); or iii) a sequence selected from SEQ ID NOS: 50-67. 6. The method according to p. The method of any of claims 3, 4 or 5, characterized in that groups of low and high risk HPV genotypes are detected. 7. The method according to p. The method of any of the preceding claims, further comprising detecting the synthetic oligonucleotide as an internal standard. 8. The method according to p. The method of claim 7, wherein the internal template is the sequence of SEQ ID NO: 68 and for detecting it, SEQ ID NO: 69 is used as hybridization probe. PL 209 415 B1 9. A kit of reagents for the detection and typing of HPV, characterized in that it comprises: i) a primer mixture as defined in claim 1 1; ii) optionally an oligonucleotide as internal standard, preferably of SEQ ID NO: 68; iii) hybridization probes containing or hybridizing to SEQ ID NO: 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109 or 111 and possibly with the sequence of SEQ ID NO: 76, 78, 80, 82, 84, 86, 88 , 90, 92, 94, 96, 98, 100, 102, 104, 106, 108 or 110; and iv) optionally, hybridization probes, preferably of SEQ ID NO: 69, detecting the pattern of